Biology subjects

Keating, M.

Publications and source records attributed to Keating, M..

4 recordsLinked to original sources

bioRxiv2026

Pax9 governs anterior identity and deployment of sclerotome to the median fins

The caudal fin is an anomaly among vertebrate locomotory appendages: its internal skeleton is as asymmetric as the human hand, but it lacks the Shh-secreting Zone of Polarizing Activity (ZPA) and the Gli3/HoxD/Hand2 programs that govern anterior-posterior patterning in other appendages. As the caudal fin was also the first appendage to evolve, deciphering its alternative patterning program may provide clues to the ancestral state. Pax9 is one of few conserved appendage patterning factors reported to also be active in the caudal fin, specifically in the anterior domain. We report that loss of pax9 function in zebrafish not only disrupts anterior-specific caudal fin anatomy, but also results in a spectacular fusion of the caudal and anal fins along the ventral midline. The dorsal fin is also expanded to a lesser degree, the paired fins not at all. The mutant caudal fin initially forms as an irregularly patterned structure lacking anterior molecular identity, with supernumerary elements spilling out beyond the normal anterior boundary. Unexpectedly, this phenotype is subsequently compounded by neighboring trunk somites erroneously deploying skeletal mesenchyme in the normally finless caudal peduncle region, completing the ectopic skeleton. scRNAseq analysis at caudal fin bud stage indicates that pax9 mutants gain skeletal mesenchyme at the expense of a specialized type of fibroblast involved in the formation of fin fold actinotrichia. Median fin skeletal mesenchyme and fin fold fibroblasts both arise from the sclerotome, a somite compartment that also robustly expresses pax9. We propose that, within the sclerotome, Pax9 pushes progenitors towards fin fold fibroblast fate, limiting how many cells will later be available to make median fin skeleton. Within the fin bud, it drives anterior identity, with the strongest impact on the ZPA-free caudal fin bud. These dual sites of action make Pax9 a uniquely powerful governor of median fin development.

Developmental Biology↗

bioRxiv2026

Pupil-DLC: an open-source deep learning pipeline for scalable, markerless tracking of pupil dynamics across conscious and unconscious states

BackgroundPupil diameter is a non-invasive biomarker of brain state, correlating with arousal, attention, cognitive processing, and consciousness. However, existing pupillometry software often lacks scalability and robustness across diverse experimental conditions and species. New methodWe introduce Pupil-DLC, an open-source, offline, DeepLabCut-based pipeline for scalable, marker-less pupil tracking, primarily designed for mice. Trained on 21,750 manually annotated frames from over 140 videos of head-fixed mice spanning wakefulness and drug-induced states, including psychedelics and anesthesia, the dataset was deliberately selected to maximize pupil size variability and model generalization. Pupil-DLC implements a dual-model architecture: a General Model (GM) for high-throughput analysis and an Individual Model (IM) for session-specific optimization. ResultsPupil-DLC captures pupil dynamics across awake, psychedelic, and anesthetized conditions with high agreement with ground truth and equal tracking fidelity during active locomotion and quiet rest. Confidence metrics aligned with human frame quality assessments, enabling principled tuning of accuracy-retention trade-offs. As a secondary demonstration, Pupil-DLC extends to unseen human videos across diverse conditions and frame rates, including daylight and smartphone recordings, without retraining. Comparison with existing methodsPupil-DLC outperforms existing automated methods in accuracy and frame retention while maintaining computational efficiency comparable to real-time tools. These improvements stem from a learned keypoint-based representation robust to pupil shape variability, occlusions, reflections, and imaging artifacts. The GM/IM framework supports a tiered strategy balancing throughput and precision. ConclusionsPupil-DLC provides a reproducible, adaptable platform for quantifying pupil-linked brain state dynamics across experimental paradigms and species, bridging basic mouse neuroscience and translational human applications.

neuroscience↗

bioRxiv2025

Protein Secretion From Liver Ex Vivo Reveals Regulation of Hepatokines by Sex and Circadian Rhythm

Here, we use an ex vivo approach compatible with the circadian timescale to interrogate protein secretion from liver, revealing several findings. Proteomic analyses in male and female mice identify hundreds of proteins that exhibit time-of-day-dependent or clock-dependent secretion involved in extracellular matrix, immune, redox, xenobiotic, and fatty acid functions. Among these, the liver secretes more endostatin, a cleavage product of collagen type XVIII alpha 1 (COL18A1), during the inactive, fasting phase of the diurnal cycle. Temporal regulation of COL18A1/endostatin is dysregulated upon loss of Bmal1 through combined effects on Col18a1 transcriptional repression and proteolytic processing. Functional experiments in vivo and in vitro reveal that endostatin suppresses mitochondrial gene expression in white adipose tissue in a time-dependent manner and reduces mitochondrial respiration in adipocytes, while enhancing lipolysis. These results support a mechanism of inter-organ crosstalk whereby hepatically derived, temporally-restricted endostatin tunes adipocytes toward metabolic activities required during the fasting phase.

molecular biology↗

bioRxiv2023

A robust knock-in approach using a minimal promoter and a minicircle

Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.

developmental biology↗